Coumarin linked to 2-phenylbenzimidazole derivatives as potent α-glucosidase inhibitors

α-Glucosidase inhibitors have emerged as crucial agents in the management of type 2 diabetes mellitus. In the present study, a new series of coumarin-linked 2-phenylbenzimidazole derivatives 5a–m was designed, synthesized, and evaluated as anti-α-glucosidase agents. Among these derivatives, compound 5k (IC50 = 10.8 µM) exhibited a significant inhibitory activity in comparison to the positive control acarbose (IC50 = 750.0 µM). Through kinetic analysis, it was revealed that compound 5k exhibited a competitive inhibition pattern against α-glucosidase. To gain insights into the interactions between the title compounds and α-glucosidase molecular docking was employed. The obtained results highlighted crucial interactions that contribute to the inhibitory activities of the compounds against α-glucosidase. These derivatives show immense potential as promising starting points for developing novel α-glucosidase inhibitors.

In view of the mentioned scaffolds, herein, by connection of coumarin to 2-phenylbenzimidazole by carboxylate linker, a novel series of α-glucosidase inhibitors was designed.Next, all designed compounds were examined against yeast α-glucosidase.Finally, in silico assessments were done to get insight into the structure-activity relationships (SARs) and binding interaction modes of the title compounds.

In vitro α-glucosidase inhibition and SAR discussion
The potency of the target compounds 5a-m was evaluated in vitro against α-glucosidase, compared with acarbose used as a positive control.The IC 50 values obtained for each compound are listed in Table 1.Notably, compound 5k, which contains a 2-hydroxyphenyl moiety, exhibited promising activity with an IC 50 value of 10.8 ± 0.1 µM, making it the most potent inhibitor compared to acarbose.
In the present study, compound 5a, an un-substituted derivative (R=H), demonstrated an IC 50 value of 50.0 ± 0.8 µM, which was approximately 15-fold better than acarbose.
Substituting chlorine at the ortho position (5e; IC 50 = 34.7 ± 0.7 µM) or bromine at the meta position (5f.; IC 50 = 34.7 ± 0.7 µM) resulted in reduced activity compared to their para-substituted counterparts.However, despite the decrease in activity, these derivatives still exhibited better inhibition compared to un-substituted derivative 5a.
Furthermore, it was observed that the introduction of second fluorine atom on 4-fluorophenyl ring of compound 5b and the second chlorine atom on 2-chlorophenyl ring of derivative 5e, as in compounds 5g and 5h, respectively, reduced anti-α-glucosidase activity.
SAR study also demonstrated that electron-withdrawing groups at the para position of the phenyl ring are more effective than electron-donating groups (halogen derivatives 5b-d vs. methyl and methoxy derivatives 5i-j).In contrast, 2-hydroxy derivative 5k was significantly more active than 2-chlorophenyl derivative 5e.
Moreover, SAR study of compounds with two deference substitutions, involving both electron-donating and electron-withdrawing groups, was performed.The lowest potency was observed in 2-CH 3 -3-NO 2 derivative 5l, with an IC 50 value of 119.5 ± 0.1 µM.Replacing of 3-NO 2 group of compound 5l with 3-Cl substituent, as in compound 5m, improved the potency.
In addition to the listed of IC 50 values in Table 1, for example, two dose response curves for two compounds 5c and 5d were showed in Fig. 2.

Enzyme kinetic studies
According to Fig. 3a, the Lineweaver-Burk plot showed that the Michaelis-Menten constant (K m ) gradually increased and maximum velocity (V max ) remained unchanged with increasing inhibitor concentration indicating a competitive inhibition.The results show 5k binds to the active site on the enzyme and compete with the substrate for binding to the active site.Furthermore, the plot of the K m versus different concentrations of inhibitor gave an estimate of the inhibition constant (K i ) of 10.4 µM (Fig. 3b).

Molecular docking
In the enzyme assay section, it was reported that the assay was conducted utilizing the enzyme Saccharomyces cerevisiae α-glucosidase (EC. 3. 2. 1. 20).However, due to the unavailability of the 3D crystallographic structure of this enzyme in the corresponding databases, a new structure was developed using homology modeling 33 .
After that, based on obtained mode of representative compound (the most potent compound 5k) in the kinetic study (competitive mode), docking study of the target compounds was performed in the α-glucosidase active site.Superimposed structure of the standard inhibitor (acarbose, pink) and the most potent compound 5k (orange) is showed in Fig. 4a.as can be seen in Fig. 4b, acarbose as positive control established hydrogen bonds with residues Thr307, Thr301, Asn241, Glu304, Ser308, Phe157, and Pro309, Arg312, and Gln322, non-classical hydrogen bonds with residues Val305, His239, Arg312, and Glu304, a hydrophobic interaction with His279, and two unfavorable interactions with residues Thr307 and Arg312 in the α-glucosidase active site.
For this study, we considered the compounds 5c-e, 5k, and 5l-m because they were either potent or had interesting points in term of SARs.Interaction modes of compounds 5c-e, 5k, and 5l-m were showed in Figs. 5  and 6 and binding energies of these compounds and acarbose were listed in Table 2.
The comparison of binding energies of the compounds 5c-e, 5k, and 5l-m with acarbose revealed that these new compounds can be bind to the active site easier than the standard inhibitor (Table 2).These results were confirmed by the obtained results of in vitro assessment.
Interaction mode of the most potent compound 5k showed that this compound established three hydrogen bonds with residues Thr307, Thr301 (via 2-hydroxy group), and Glu304 (Fig. 5).This compound also formed a π-anion interaction with Glu304 and a π-cation interaction with His239.Moreover, hydrophobic interactions  www.nature.com/scientificreports/ between compound 5k and residues Glu304, His239, Val305, Pro309, and Arg312 were observed.In vitro study demonstrated that compound 5k with 2-hydroxy substituent was twofold more potent thah the second potent compound 5d with 4-bromo substituent (Table 1).The comparison of binding energies of these compounds showed that compound 5k had lower binding energy in comparison to compound 5d.Compound 5d formed four classical hydrogen bonds with residues Asn241, Asn412, Ser308, and His239 and a non-classical hydrogen bond with His279 (Fig. 5).Glu304 established a π-anion interaction with compound 5d.Furthermore, the latter compound created hydrophobic interactions with residues Arg312 and Pro309.Replacement of 4-bromo substituent of compound 5d with 4-chloro substituent, in case of compound 5c, led to a negligible decrease in inhibitory activity and a negligible increase in binding energy (Table 1 and 2).As can be seen in Fig. 5, 4-chloro derivative 5c formed three classical hydrogen bonds (with Asn241, His239, and Ser308) and a nonclassical hydrogen bond (with Arg312) with the active site of α-glucosidase.This compound established π-anion interactions with Glu304 and hydrophobic interactions with Arg312, Pro309, and Val305.The comparison of IC 50 values of 4-chloro derivative 5c and 2-chloro derivatives 5e demonstrated that translocation of chlorine atom on the 2-phenyl ring had a moderate effect on anti-α-glucosidase activity, and 4-chloro was preferred (Table 1).
The obtained binding energies of compounds 5c and 5e were in agreement with in vitro study (Table 2).The comparison of binding modes of the latter compounds showed that 4-chloro derivative established three classical hydrogen bonds and a non-classical hydrogen bond while 2-chloro derivative formed two classical hydrogen bonds with Gly306 and Arg312 and two non-classical hydrogen bonds with His239 and Pro309.According to the mode of interaction of acarbose (Fig. 4b), it seems that Gly306 is not very important in the occurrence of the effect.π-Anion interactions were similar in both of compounds 5c and 5e.Moreover, hydrophobic interactions also were similar in these compound only compound 5e interacted with Phe311 while compound 5c interacted Arg312.

Conclusion
Following our expertise in the rational design of α-glucosidase inhibitors, a series of coumarin-benzimidazole derivatives were designed and synthesized.The chemical structures of these derivatives were thoroughly characterized using various analytical techniques, including 1 H-NMR, 13 C-NMR, and FTIR analysis.Our results revealed that all the synthesized compounds exhibited significant anti-α-glucosidase activity, as demonstrated by their IC 50 values ranging from 10.8 ± 0.1 μM to 119.5 ± 0.1 μM, in comparison to the reference compound acarbose (IC 50 = 750.0µM).These findings highlight the efficacy of the designed backbone in targeting α-glucosidase.Among the tested compounds, compound 5k emerged as the most potent inhibitor with an IC 50 value of 10.8 ± 0.1 μM.Further kinetic experiments revealed that compound 5k exhibited a competitive inhibition pattern.To gain deeper insights into the interaction mechanism of these derivatives at the α-glucosidase active site, we performed molecular docking study.By this study, the observed SARs were rationalized and possible binding modes were detected.

Figure 1 .
Figure 1.Rationale for the design of coumarin linked to 2-phenylbenzimidazole derivatives as the new α-glucosidase inhibitors.

Figure 3 .
Figure 3. Kinetics of α-glucosidase inhibition by 5k (inhibitor): (a) the Lineweaver-Burk plot in the absence and presence of different concentrations of 5k; (b) the secondary plot between K m and various concentrations of 5k.

Figure 5 .
Figure 5. Interaction modes of compounds 5c-e and 5k in the α-glucosidase active site.

Figure 6 .
Figure 6.Interaction modes of compounds 5m and 5l in the α-glucosidase active site.

Table 1 .
α-Glucosidase inhibition assay of the target compounds 5a-m.a Data represented in terms of mean ± SD. b Positive control.